Expression of Viral Capsid Protein as a Potential Vaccine Candidate for Rotavirus

FEATURED CASE STUDY

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Background:

Viral capsid glycoprotein X (Due to confidentiality, we are not disclosing the information about protein) is the primary candidate for inclusion in a subunit vaccine against rotaviral diarrhea. Therefore, significant effort has been directed towards producing recombinant protein X that maintains the antigenic characteristics of the native molecule. A successful vaccine that will induce adequate neutralizing antibody will need to present protein X in a conformation similar to the mature virus.

Problem/Rational:

Previous literature suggests that the expression of protein X in other expression platforms (E. coli, baculovirus) leads to misfolding and loss of its conformation. Therefore, a selection of a suitable host is necessary. The client had tried the expression of protein X in bacterial platforms, but the protein activity was not enough to proceed further with the rest of the experiments.

Solution/Approach:

To solve this problem, we thoroughly studied the protein. We found that the presence of cysteine in the neutralization domain of glycoprotein X plays a significant role in acquiring its correct conformation. Therefore, we used our proprietary host expression platform, D-Crypt™, an engineered protease deficient yeast host cell with disrupted endogenous genes. These platform features provide an ideal eukaryotic environment for expressing recombinant proteins retaining their correct structure and function.

Briefly, the gene was cloned into proprietary expression plasmid pYRE100 in such a way that the expressed protein has a C-terminus His tag. The construct was then transformed into a proprietary protease deficient S. cerevisiae host strain named PYPD. Expression of His tag-glycoprotein was confirmed by using the anti-His antibody in Immunoblot analysis. Studies have shown that the presence of cysteine in the neutralization domain of glycoprotein X plays a significant role in enhancing vaccine efficacy. Sequencing of glycoprotein X confirmed the presence of 12 cysteine residues in its neutralization domain and, therefore, showed better activity (Figure 1: left panel) than standard glycoprotein at different dilutions of binding antibody (Figure 1: right panel) due to its correct conformation. These results suggest that a suitable host mimicking human's posttranslational modification and high purity of the protein is necessary to increase the activity of glycoprotein X.

These results further suggest the applicability of the D-crypt™ platform in increasing the expression and purity of membrane-bound proteins where posttranslational modification plays a major role.

Fig 1: ELISA demonstrating activity of rotavirus glycoprotein

Measurable impact:

1) Expression of functionally active glycoprotein having 12 cysteine residues with an appropriate folding.
2) Testing of functional activity of glycoprotein by ELISA demonstrated to be better than standard glycoprotein.

Expression of Highly Active Protein in E. coli for Diagnostics Applicatio

The production of recombinant enzymes has led to the development of many diagnostics strategies in various medical fields. The increasing demand for recombinant proteins has created the need to develop efficient protein expression techniques to increase the productivity of the product.

Expression of Nav1.7

Voltage-gated sodium channels are transmembrane proteins responsible for propagating the electrical signal in neurons, cardiac, skeletal muscle, and neuroendocrine cells. Sodium channel Nav1.7 is preferentially expressed in peripheral neurons. Nav1.7 is a validated target for pain treatment in humans. The principal subunit of this channel is a protein of >200 kDa, the alpha subunit, which consists of four large domains of internal homology. The subunit forms the pore and functions in voltage-dependent activation and inactivation,

Expression of Full-Length Neuraminidase (NA)

Below is the case study describing the expression of full-length neuraminidase in the protease-deficient strain of Baker’s yeast, S. cerevisiae of D-Crypt™. The platform provides an ideal eukaryotic environment for expressing recombinant proteins retaining their correct structure, function, and post-translational modifications resembling those of humans. We used an array of proprietary expression vectors with designed upstream sequences for enhanced expression of neuraminidase.